Direct PCR Sequencing of BS Products - Having trouble (Apr/07/2011 )
So after months and months of trying to get this to work, I have finally reached the sequencing stage.. and i'm wondering how to do this..
I've sequenced the product using my forward primer but it doesn't seem to match up with my genomic sequence (bs treated invivo).. I've used Bi-q analyzer..any thoughts on this?
It doesnt match the expected PCR sequence product that methyl primer express gave me when I was designing my primers.
How can you validate that BS sequence amplification is in the right spot corresponding to the genomic reference sequence?
There is a possibility that the product you obtained is not specific or the band you isolated contained not just one product. You really need to separate your PCR product very well to get pure population of amplicons.
If you directly sequence your product without cloning, reverse primer usually yields better sequencing results than forward primer. If you are sure your PCR product is clean, try rerun the sequencing reaction using the reverse primer.